Apparatus and methods for forming microcultures within porous media

ABSTRACT

Highly porous, beads are comprised of a polymer and a second compound mixed into it. The second compound, an amendment, is either a nutrient or a compound having high affinity to one or more nutrients. A plurality of these beads may be exposed to an aqueous environment, usually a body of water. Bacteria and other microorganisms rapidly enter and remain within the nutrient filled interior space of the beads. Any of a number of various detection methods may then be used to characterize, detect and/or identify the microorganisms.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation-in-Part of U.S. patent application Ser. No.09/728,896 filed Dec. 4, 2000 which claims priority to U.S. ProvisionalApplication Ser. No. 60/168,484 filed on Dec. 2, 1999.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to methods and devices for formingmicrocultures within porous media. Specifically, the present inventionrelates to methods and materials for: 1) capturing, collecting, orisolating various bacteria and other microorganisms from aqueousenvironments in the form of biofilms for the purpose of characterizationof community structure or identification of specific organisms or 2)concentrating and/or immobilizing specific types of bacteria and/orother microorganisms for the purpose of holding or retaining same withina bioreactor environment. The present invention may be used to capture,collect, or isolate bacteria and other microorganisms in the form ofrepresentative biofilms from water sources, such as streams, lakes,reservoirs, and groundwater as well as other aqueous environments suchas drinking water or wastewater treatment systems, cooling towers,storage tanks, or any commercial or industrial process which employs orproduces an aqueous phase. The present invention may also be used toconcentrate and/or immobilize selected bacteria or other microorganismswithin a bioreactor and thereby increase the volumetric productivity ofthe bioreactor. The present invention is a significant improvement overexisting technology in that 1) biofilms form rapidly in the presentinvention and biofilms are more indicative of the operative microbiologyof the aqueous environment to which the invention is exposed and 2) thepresent invention can also make the concentrated or immobilized statethe preferred state over the planktonic or “free-floating” mode ofexistence for specific microorganisms in a bioreactor environment,thereby concentrating and retaining specific types of microorganismswithin a bioreactor and thereby concentrating the biocatalytic activityof same and improving the performance of the bioreactor.

2. Prior Art

For a variety of reasons, it is highly desirous to have efficient,accurate, and sensitive methods of detecting, characterizing, and/oridentifying microorganisms from aqueous environments. Water treatmentfacilities for preparing potable water, sewage and wastewater treatmentfacilities, environmental engineers, ecologists, biologists, and theagriculture industry, to name a few, all require a fast and accuratemethod for determining the characteristics of operative microbialcommunities and/or the presence of specific types of microorganisms inaqueous systems.

Microorganisms in aqueous environments have a natural affinity for solidsurfaces and commonly form biofilms with complex community structures.Biofilms can concentrate nutrients, exclude toxic substances, facilitatebeneficial cross feeding, and promote other interactions betweenmicroorganisms that benefit the members of the community. Therefore,microorganisms generally prefer biofilms over the planktonic or“free-floating” state. Planktonic microorganisms in an aqueousenvironment are generally those that have not yet been taken up bybiofilms or sloughed off from biofilms as the biofilms grow in size. Ithas been frequently observed that the physiology of many microorganismsis different in biofilms compared to planktonic or “free-floating”organisms. Therefore, the microbial ecology of an environment populatedwith microorganisms is best represented by biofilm communities ratherthan by organisms that are planktonic or “free-floating”. The mostcommon current method for detecting the presence and types of bacteria,fungi, and other microorganisms in an aqueous environment consists ofplacing a series of “coupons” at various points in the system. Couponsare generally relatively small plastic strips to which bacteria andother microorganisms attach to form biofilms. Typically, these couponsmust remain in a body of water for a long period of time in order tohave an adequate amount of microbial biomass attach to facilitateanalysis of the biofilm community. The time required to form biofilmsdepends on the quality of the water and a number of environmentalfactors but incubation times of weeks or months are not uncommon. Shearforces and the lack of any attractants make coupons a relatively poorsubstrate for microorganisms to attach to. Another method of collectingrepresentative biofilm communities and detecting the presence ofspecific types of microorganisms consists of placing glass wool in aperforated vial and placing the vial in the water. However, these glasswool devices suffer from the same slow and low uptake as the moreconventional coupons.

It is therefore desirable to provide a method and device for rapiduptake of large amounts of microbial biomass in representative biofilmsfrom a variety of aqueous environments.

SUMMARY OF THE INVENTION

In the present invention, porous media are utilized to collectmicroorganisms from a surrounding aqueous phase in the form ofrepresentative biofilms; that is, biofilms that are representative ofthe biofilm communities found to exist on solid surfaces in contact withthat aqueous phase. In the preferred embodiment of the current inventionthe porous medium utilized is in the form of porous beads. The porousbeads are held within a vessel made of materials that are sufficientlychemically inert so as not to interfere with the collection ofmicroorganisms by the porous beads. The vessel containing the porousbeads is then held in contact with an aqueous phase by a variety ofmethods depending on a number of factors including whether the aqueousphase is stagnant or flowing, physical location, etc. The presentinvention is especially useful in detecting specific types ofmicroorganisms such as pathogens in water supplies. It is also usefulfor detecting entrance points of various microbes into moving bodies ofwater such as rivers and streams as well as characterizingmicroorganisms in pristine or contaminated aqueous streams such asgroundwater, drinking water, or wastewater. Further, the presentinvention is also useful for the evaluation of remediation amendmentsfor contaminated aquifers.

The preferred embodiment of the present invention is comprised of beadsapproximately 2-4 millimeters or less in diameter. These beads areformed by known techniques such that they are highly porous. To thepolymer matrix that forms the beads are added various amendments, orchemical compounds (either synthetic or naturally occurring), thateither attract and bind to nutrients from an aqueous environment, arenutrients themselves, or interact with the aqueous environment in someother way to make the inside of the beads more conducive to the growthof specific types of microorganisms. These amendments, or materialsbound to them, serve as attractants, because they attract specific typesof microorganisms either by providing nutrients preferred by specifictypes of microorganisms or providing a growth environment preferred byspecific types of microorganisms. Both the nature of the beads and thepresence of attractants cause bacteria and other microorganisms to enterthe pores in the skin of the beads, encounter copious attractive surfacearea, and thereby form biofilms or microcultures.

Within a very short amount of time, significantly faster than otherknown methods, a number of microorganisms sufficient to perform knowndetection, characterization, and identification assays are presentwithin a collection of microculture beads. These assays may be rapidlyand readily performed on the beads such that the microorganisms presentin biofilms associate with a given body of water may be accuratelydetected.

Another significant advantage of forming microcultures or biofilmswithin the beads disclosed herein is that microorganisms that aredifficult to culture in the laboratory may be readily collected becauseof the favorable growth environment provided by the biofilm communitystructures. It is well known in the art that there are a wide variety ofmicroorganisms for which no growth medium has been developed. Becausethese microorganisms cannot be grown in a laboratory, it is oftendifficult to acquire a sufficient number of required organisms in orderto reliably and accurately perform a detection, characterization, oridentification assay. The microculture beads of the present inventionovercome this deficiency by rapidly forming natural biofilmsrepresentative of the aqueous environment in contact with the beads.

Those skilled in the art will appreciate that the present invention issuitable for use with a variety of detection, characterization, andidentification assays. Phospholipid fatty acid (PLFA) analysis,respiratory quinone analysis, polymerase chain reaction (PCR)amplification of 16S rDNA, real-time PCR, and other techniques ofmolecular biology may all be utilized to detect, characterize, and/oridentify microorganisms in the present invention without the need ofremoving microorganisms from the beads. Another advantage of the currentinvention is the observation that biomolecules, especially DNA, is morereadily extracted (faster, more efficiently, and with lesscontamination) from the current invention than environmental samples.

It is therefore an object of the present invention to provide beadscapable of forming representative microcultures or biofilms ofmicroorganisms from aqueous phases within the beads.

It is another object of the present invention to provide a method forcharacterizing, detecting, and identifying the presence of specifictypes of microorganisms in an aqueous solution or body of water.

It is another object of the present invention to provide a method ofculturing microorganisms that are unsuitable for culturing on agarplates or liquid media.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph of the ability of the porous beads of the presentinvention to collect biomass in a drinking water system compared to aconventional PVC coupon

FIG. 2 is a graph of the ability of the porous beads of the presentinvention to collect biomass in a PCE contaminated aquifer compared toglass wool

FIG. 3 is a scanning electron micrograph of a cross section of a porousbead of the present invention

FIG. 4 contains two high-magnification scanning electron micrographs ofthe skin of the porous beads of the present invention

FIG. 5 is a high-magnification scanning electron micrograph of theinterior of a porous bead of the present invention

FIG. 6 is a schematic diagram of an interior surface of the porous beadsof the present invention

FIG. 7 is a schematic diagram of a device for collecting representativebiofilms from water using porous beads of the current invention

FIG. 8 is a scanning electron micrograph of a PAC/HRC bead

FIG. 9 shows the biomass PLFA collected by PAC and PAC/HRC beads in anaquifer contaminated with PCE

FIG. 10 shows a relationship tree comparing PLFA collected by PAC andPAC/HRC beads in an aquifer contaminated with PCE

FIG. 11 a,b shows the respiratory quinones identified in PAC and PAC/HRCbeads in an aquifer contaminated with PCE

FIG. 12 compares the Shannon diversity index for PLFA and respiratoryquinines from biomass collected by PAC and PAC/HRC beads in an aquifercontaminated with PCE

FIG. 13 is a DGGE gel image comparing 16S rDNA segments obtained fromPAC and PAC/HRC beads in an aquifer contaminated with PCE

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The embodiments discussed herein are merely illustrative of specificmanners in which to make and use the invention and are not to beinterpreted as limiting the scope of the instant invention.

While the invention has been described with a certain degree ofparticularity, it is to be noted that many modifications may be made inthe details of the invention's construction and the arrangement of itscomponents without departing from the spirit and scope of thisdisclosure. It is understood that the invention is not limited to theembodiments set forth herein for purposes of exemplification.

In the present invention, highly porous beads are formed having veryinternal surface area. The beads used herein are very similar to theporous beads described in U.S. Pat. No. 5,486,292 to Bair owned by theAssignee herein, which is incorporated herein by reference. The beads ofthe present invention are preferably comprised of an aramid polymerselected from poly(m-phenylene isophthalamide) and copolymers thereof,and a polymer or copolymer formed from m-phenylene diamine and an acidselected from the group consisting of terephthalic acid, isophthalicacid and 2,4-diaminobenzene sulfonic acid. It is also preferable toutilize beads made from polymers capable of withstanding sterilizationand cleaning methods, such as high temperature (300° C.) and thecombination of high temperature and pressure created by autoclaves. Moresensitive plastics may be used. However, because these plastics cannotbe sterilized, they are practically guaranteed to contain viablemicroorganisms and have residual “fossil” biomarkers in them that willresult in false readings of microorganisms characterization, detection,and identification. Unsterilized beads may also have DNAse's and RNAse'sthat can frustrate hybridization and PCR procedures.

The microculture beads of the present invention are comprised of apolymer as described above and additionally one or more amendments whichmay or not include powdered activated carbon (PAC) which is an integralcomponent of the beads in the patent referenced above. The amendments ofthe present invention are either nutrients themselves, are capable ofbinding nutrients from the aqueous environment the beads are in contactwith, or capable of interacting with the internal bead environment toproduce conditions more favorable to microbial growth. These amendmentsand/or materials bound to them serve as both attractants and a foodsource for specific microorganisms or modifiers of the bead environment.These beads are generally placed in a water permeable vial or vessel andimmersed in the body of water to be tested. Bacteria and othermicroorganisms are attracted to the internal surface of the beads andrapidly form microcultures or biofilms. Factors which account for therapid formation of biofilms in the beads include hydrophobicity of thepolymer surface, high internal surface area (>200 m² per gram), lowshear conditions, concentration of limiting nutrients by the PAC orother amendments, and rapid formation of pre-conditioning films. It iswell know that the rate-limiting factor in the formation of naturalbiofilms is the availability of some limiting nutrient, usually a carbonsource. PAC and other amendments bind and concentrate nutrients on theinside of the beads greatly favoring biofilm formation inside the beads.A wide variety of methods in molecular biology for characterization,detection, and identification of microorganisms known to those skilledin the art may then be applied to the beads in order to determine thepresence and identity of the various microorganisms formingmicrocultures or biofilms within the beads.

Formations of microcultures within the beads is extremely fast comparedto existing methodologies. For example, a vial of about 500 beads iscapable of producing enough organisms in microcultures or biofilms indrinking water systems to perform detection assays within a 24-hourperiod. This is significantly shorter than the one-month collection timerequired by coupons in drinking water systems as shown in FIG. 1. Inaddition, as shown in FIG. 2, the amount of microbial biomass collectedby a vial of about 50 beads in contact with an aquifer contaminated withan average of 2 mg/L of perchloroethylene (PCE) for 30 days was sixtimes greater than that collected an equal volume of glass wool over thesame time period. In each case the attractant was PAC.

A scanning electron micrograph (SEM) of a typical porous bead of thepresent invention is provided in FIG. 3. The highly porous nature of thebeads is evident in this micrograph. The porous bead is typically 2 to 4millimeters in diameter. It is preferable to have beads of relativelyuniform size. Having a mixture of beads wherein some of the beads aremany times smaller than the largest beads is generally undesirable, asthe smaller beads may become lodged in the spaces between the largerbeads and reduces the productivity of the bead vial or vessel bylimiting the movement of water within the vial. FIG. 4 provideshigh-magnification SEMs of the skin of the porous beads showing theholes and tears in the skin which provide access for microorganisms tothe inside of the beads. FIG. 5 provides a high-magnification SEM ofsome of the interior surface of the beads available for the formation ofmicrocultures or biofilms. FIG. 6 is a schematic diagram of a typicalinterior surface of the beads that form the present invention. The beadsinterior surfaces 10 are comprised of polymer 14 and amendments 16. Thepolymers mentioned above are generally the most preferable polymersused. However, any polymers that are capable of withstanding the hightemperatures of sterilization and cleaning techniques and susceptible totechniques of forming porous beads when mixed with some type ofaggregate such as the amendments listed herein will be suitable.Amendments 16 may be any of a wide variety of materials. These materialsfall into one of two categories, and some of the amendments overlap intoboth categories.

The first category of amendments is materials that may be used to bindnutrients. Amendments 16 in FIG. 6 are of this variety. Activatedalumina, silica gel, certain clays, adsorptive resins, and zinc oxideare examples of amendment materials in this category. In addition tothese and similar amendments PAC may also be incorporated into thepolymer matrix as described by U.S. Pat. No. 5,486,292 to Bair owned bythe Assignee and referenced above. Powdered activated carbon (PAC) bindsto a wide variety of nonpolar organic compounds including hydrocarbonsand halogenated hydrocarbons. PAC may also be employed in fabrication ofthe porous beads of the present invention strictly to produce desiredphysical properties in the beads. Activate alumina and silica gel bindcertain classes of polar organic compounds, clays bind certain cations,zinc oxide binds well to sulfides and mercaptans, whereas adsorptiveresins can other similar materials can be fashioned to have even morespecific adsorptive properties. Referring to FIG. 6, amendments 16 areinterspersed throughout the porous bead of the present invention. Theymay have nutrients 18 bound to them on portions of the surface areawhere the amendments are exposed.

There are two methods by which nutrients may be applied to theamendments within the beads. One option is to treat the porous beadsafter they have been cleaned and sterilized but before they are exposedto the aqueous environment to be tested. Treatment consists of soakingthem in a solution having a relatively high concentration of thenutrients that are desired to be attached to the amendments in thebeads. The nutrients will bind to the amendments, and the beads may thenbe placed in a vial, vessel or other suitable device for holding them.The vial of beads is then introduced to the aqueous environment to betested. The second method of attaching nutrients to the amendments is toexpose the aqueous environment to be tested subsequent to cleaning andsterilization. Nutrients in the aqueous environment may then naturallyattach to the porous amendments in the porous beads therebyconcentrating the nutrients on the inside of the beads.

Microorganisms will be attracted to nutrients inside the beads andwithin the macro and micro pores of the beads. Once nutrients attachedto amendments are depleted by consumption by microorganisms, othernutrients in the aqueous environment will then bind to the amendmentssuch that microorganisms are provided a constant, steady supply ofnutrients on which to feed. The second class of amendments are thosethat are themselves nutrients. Those skilled in the art will appreciatethat there are a large variety of compounds that make suitablenutrients. In addition, selecting particular nutrients will allowselectivity of the porous beads for a class of microorganisms, or even aspecific microorganism. For example, utilizing elemental sulfur as theamendment will result in cultures comprised primarily of sulfotrophs.Organisms that do not metabolize sulfur will be less attracted to theporous bead internal environment. Other compounds that may be used asamendments that serve as attractive nutrients include polylactates,known commercially as hydrogen release compound (HRC), which slowlyhydrolyze to generate lactate and carbonates (for autotrophs).Alternatively, to ensure that all desired types of microorganisms arecaptured by the porous beads and propagated to a level that allowscharacterization, detection, and/or identification a mixture of severaldifferent nutrients may be used as amendments incorporated into theporous beads. In addition porous beads of the current invention mayincorporate both types of amendments described herein. These types ofamendments which are themselves nutrients are not illustrated in FIG. 6but the difference is subtle. Referring to FIG. 6, if the amendments 16were themselves nutrients, microorganisms would be feeding directly onthem and there would be no attached nutrients 18.

FIG. 7 shows an illustrative device for use in preparing microcultureswithin the porous beads in order to characterize, detect, and/oridentify microorganisms present in a body of a water such as groundwatercollected in a groundwater monitoring well. Testing device 30 iscomprised of rope 32 to which weight 34 and vials 36 are attached. Thisdevice is especially well suited for a variety of bodies of water.Weight 34, located at the bottom of rope 32, causes the device to sinkas far down as desired. Vials 36 are separated by a distance 40. Thisdistance will be determined by the person testing the water. Thoseskilled in the art will appreciate that different types of microorganismmay exist in different concentrations at different depths within a bodyof water. The use of multiple vials allows each level of the water to betested simultaneously. How long distance 40 is will depend on a varietyof factors known to those skilled in the art. Vials 36 are attached torope 32 by attaching clamps 38. Those skilled in the art will appreciatethat there are a wide variety of means by which to attach vials to arope. Vials 36 themselves may also take any of a wide variety of shapes.Throughout this disclosure, porous bead holding devices are referred toas vials or vessels. However, those skilled in the art will appreciatethat these vials or vessels may be made of plastic, glass, metal, wood,cloth or any of a wide variety of other materials, so long as the porousbead holding vial is water permeable and prevents the porous beads fromleaving the vial or vessel. Therefore, if a porous material is used, thepores of the material must be substantially smaller than the microbeadsbut also large enough for water and microorganisms to enter the vial.Those skilled in the art will appreciate that there are a variety ofsuitable materials for making porous bead vials.

The device in FIG. 7 is suitable for use in lakes, reservoirs, wells,rivers and streams, and aquifers. In the case of rivers or streams, itmaybe desirable to have only a single vial on device 30 and to tie rope32 to an object near the stream. Several of these devices could beplaced along the river in order to determine where which microorganismsenter the stream. Alternatively, device 30 may have several vialsattached for use in a deep lake, reservoir or even water well.

Once sufficient time has been given for microorganism collection andpropagation, porous beads may be removed from the tested water and betreated by any of a variety of detection methods in order tocharacterize, detect, and/or identify the microorganisms present in thebody of water. The period of incubation of the porous beads vials orvessels in the aqueous phase to be tested will be variable depending onwater quality and environmental conditions; however, incubation times of24 hours or less may be suitable.

This method of collecting microorganisms is especially advantageous inthat it does not require growth on an agar or liquid medium in alaboratory. There are a number of microorganisms for which agar andliquid growth medium are lacking. The use of porous beads of the presentinvention circumvents that problem.

Relatively simple detection methods, such as quantifying the amount ofprotein present, may be utilized to simply test the presence ofmicroorganisms in the water. More complex tests, such as PLFA and PCRamplification of 16S rDNA may be used to characterize the community orto determine the exact identity of the microorganisms within the poriousbeads. For example, those skilled in the art will appreciate that porousbeads, may be extracted with chloroform/methanol solvents to recoverDNA. Appropriate primers can then be used to amplify 16S rDNA.Denaturing gradient gel electrophoresis (DGGE) may then be used toseparate 16S rDNAs from different species of eubacteria, archaea, orfungi (depending on the primers used). Separated 16S rDNA segments maythen be sequenced to identify the organisms from which the genes wereisolated.

“Amendments” refers generally to any chemical compound (synthetic ornaturally occurring) added to a polymeric composition from which porousbeads are formed.

“Nutrients” and “Attractants” both refer generally to chemical compounds(synthetic or naturally occurring) or materials to which microorganismsare attracted. These are generally materials that are metabolized bymicroorganisms.

“Porous Beads” generally refers to highly porous beads of the presentinvention. These beads are generally less than 2-4 millimeters indiameter and generally have a porosity or void volume of about 70%.Although the present disclosure relates to porous beads, the inventionmay also be practiced with other porous media. The same basic methodsused to make highly porous beads may also be used to make othergeometric forms. Tubes, rods, disks, larger-scale beads, smaller-scalebeads, and other geometric forms may also be made being highly porouswith large amounts of available surface area. Although porous beads mayhave a larger surface area/g the surfaces of these other geometric formswould be substantially similar to the surfaces in the porous beadsdescribed above.

Whereas, the present invention has been described in relation to thedrawings attached hereto, it should be understood that other and furthermodifications, apart from those shown or suggested herein, may be madewithin the spirit and scope of this invention.

Illustrative Example of the Preferred Embodiment

The following is an illustrative example of the preferred embodiment ofthe present invention in which porous beads have been amended with PACand a polylactate commercial product known widely as HRC. FIG. 8provides a SEM of a cross section of the PAC and HRC amended porousbeads. These porous beads were used to evaluate the potential of HRC toalter the microbial ecology of an aquifer contaminated withperchloroethylene (PCE) to favor organisms that are capable ofreductively dechlorinating PCE and other halogenated hydrocarbons. Inthis application HRC would act as what is called a remediationamendment. Typically remediation amendments for contaminated groundwaterare tested for efficacy by pilot-scale injection of the amendment intothe aquifer followed by detailed sampling and analysis of groundwaterand soil cores to determine if the desired changes in subsurfacemicrobial ecology have been achieved. The present invention can be usedto evaluate these amendments without the cost of such an exercise bycontacting the contaminated aquifer with porous beads of two types:beads impregnated with PAC and HRC and beads impregnated with just PAC.The beads impregnated with just PAC concentrate halogenated hydrocarbonsand other carbon sources inside the beads through the attraction ofthese compounds for the activated carbon surface. This favors theenvironment inside the bead for collection and growth of microorganismscharacteristic of active biofilms in that environment. In other wordsthe PAC impregnated beads provide a “before” look at the microbialecology of the unamended contaminated subsurface. HRC has been shown tostimulate the growth of microorganisms which dechlorinate PCE and otherhalogenated hydrocarbons. However, the response is not universal. ThePAC/HRC impregnated beads will provide HRC as an additional carbon andenergy source and thus, after suitable incubation in the contaminatedaquifer, the microbial ecology of these beads will predict the effect ofthis amendment on the in situ microbial ecology of the aquifer when thismaterial is injected into the aquifer.

The following testing was conducted at the site of a former dry cleaningbusiness that had used PCE as the cleaning solvent. One groundwatermonitoring well (MW1) was installed upgradient of the source and fourgroundwater monitoring wells (MW2A, MW2B, MW3, and MW4) were installeddowngradient of the source. All wells had 10-ft screens across the watertable. PCE was detected in all but MW1 at concentrations less than 10mg/L. Products of reductive dehalogenation of PCE, namelytrichloroethylene (TCE), cis-1,2-dichloroethylene (cisDCE), and elevatedchloride ion were detected in downgradient wells indicating thatreductive dechlorination was ongoing in the contaminated aquiferalthough incomplete.

A device similar to that illustrated in FIG. 8 was used to suspend bothPAC and PAC/HRC impregnated beads in each monitoring well just below thewater table. Beads were housed in PFA tubing with 3/32-in holes to allowwater penetration. Tubing separately housing PAC beads and PAC/HRC beadswere attached to the same nylon line and spaced 1 ft apart in each well.Each sampler contained about 60 beads and all samplers were incubated incontact with groundwater for 30 days.

FIG. 9 show the total phospholipid fatty acid (PLFA) collected by eachsampler in each well on a per bead basis. The amount of PLFA collectedis proportional to the amount of viable biomass. It is seen from thisfigure that the presence of HRC greatly stimulated the collection ofbiomass in the contaminated part of the aquifer. The various types andrelative amounts of fatty acids found in the collected PLFA arecharacteristic of the microbial community structure. FIG. 10 shows arelationship analysis of the fatty acids collected from PLFA in the twotypes of beads. This figure shows that the microbial communitiescollected in the two types of beads were very different from each other,and further all the communities collected by PAC beads were similar toeach other and all the communities collected by the PAC/HRC beads weresimilar to each other in the contaminated wells. When the respiratoryquinones from the two types of beads were compared from the contaminatedwells it was seen that the distributions of different respiratoryquinones were also different (FIG. 11 a,b) further illustrating that themicrobial communities in the two types of beads were quite different.When the Shannon's diversity index was calculated for the PLFA andrespiratory quinones for the two types of beads and plotted as a scatterplot (FIG. 12) it was seen that both of these analyses indicated areduction in microbial diversity in the PAC/HRC beads compared to thePAC beads.

FIG. 13 provides a gel image of 16S rDNA genes obtained from beads ofboth types. The reduced microbial diversity indicated by the PLFA andrespiratory quinone analyses was also reflected in the 16S rDNA data.Sequencing of the indicated bands revealed isolates and clones ofbacteria previously identified in sites contaminated with halogenatedhydrocarbons in four samplers, three of those containing PAC/HRC beads.

Taken all together the molecular analyses of the two types of beadspredicts that amending the aquifer with HRC will stimulate microbialbiomass production, decrease microbial diversity, and favor growth oforganisms which metabolize halogenated hydrocarbons. Thus the sameinformation sought by pilot-scale injection of HRC, coupled withmicrobial analysis of numerous groundwater samples and sediment coreswas obtained using the current invention at a fraction of the cost.

1-20. (canceled)
 21. A method to produce and use a microculture orbiofilm to detect and identify specific organisms, which methodcomprises: preparing a plurality of beads by preheating to a sufficienttemperature such that biomarkers are destroyed, said beads having atleast one polymer and at least one amendment incorporated therein;exposing said beads to an aqueous environment of a body of water havingat least one microorganism; allowing sufficient time for said at leastone microorganism to enter into said plurality of beads; and performingat least one assay on said plurality of beads in order to characterizeand identify said at least one microorganism.
 22. A method to produceand use a microculture or biofilm to detect and identify specificmicroorganisms as set forth in claim 21 wherein said at least assay ischosen from the group consisting of phospholipid fatty acid (PLFA)analysis, respiratory quinone analysis, polymerase chain reaction (PCR)amplification of the 16SrDNA gene followed by separation and sequencingand real-time PCR.
 23. A method to produce and use a microculture orbiofilm as set forth in claim 21 including at least one nutrientattached to said amendment.
 24. A method to produce and use amicroculture or biofilm as set forth in claim 23 wherein said at leastone nutrient is selected from the group consisting of hydrocarbons,halogenated hydrocarbons, methyl-t-butyl ether and t-butyl alcohol. 25.A method to produce and use a microculture or biofilm as set forth inclaim 21 wherein each said bead has a void volume which is at leastabout 40% of a total bead volume.
 26. A method to produce and use amicroculture or biofilm as set forth in claim 21 including solventextraction of biopolymers, and molecular analysis of biopolymers.